The vesicular stomatitis virus G glycoprotein (hereinafter referred to as “VSV-G”) is widely used to pseudotype viral vectors due to its wide tropism and stability. These viral vectors facilitate gene transduction in human and animals. The VSV-G proteins, when not associated with any viral vectors, are also alone capable of forming complexes with naked plasmid DNA in cell free conditions which can be transfected to cells thereafter.
The fusogenic G glycoprotein of the vesicular stomatitis virus has proved to be a useful tool for viral-mediated gene delivery by acting as an envelope protein. Due to its wide tropism, VSV-G has been used as an efficient surrogate envelope protein to produce more stable and high titer pseudotyped murine leukemia virus (MLV)-based retrovirus and lentivirus-based vectors, all of which have been effectively used for gene therapy. The reason behind this pantropism of VSV remained elusive for a long period. Recently, it has been found that the VSV enters the cell through a highly ubiquitous low-density lipoprotein (LDL) receptor having wide distribution.
However, there are some limitations associated with the use of VSV-G. It is cytotoxic to producer cells, though the use of tetracycline-regulated promoters has helped to overcome this problem. In addition, serum inactivation of VSV-G pseudotyped viruses poses a problem and impedes their function to some extent in vivo. To overcome the latter problem, VSV-G mutants have been generated which are more thermostable as well as serum-resistant. VSV-G mutants harboring T230N+T368A or K66T+S162T+T230N+T368A mutations exhibited more resistance to serum inactivation and higher thermostability.
Apart from acting as a fusogenic envelope protein for many viral vectors, previous studies showed that purified soluble VSV-G itself can be inserted into lipid bilayers of liposomes and lipid vesicles in cell free system in vitro. Additionally, it has been shown that VSV-G can form a complex with naked plasmid DNA in the absence of any transfection reagent and can thereby enhance the transfection of naked plasmid DNA into cells. Sucrose gradient sedimentation analysis demonstrated that VSV-G associates with plasmid DNA and MLV gag-pol particles to form ternary complexes that co-sediment with high DNA transfecting activity. This transfection could be abolished by adding antibody for VSV-G.
In eukaryotic cells, heritable genetic material is packaged into structures known as chromatin consisting of DNA and protein. The basic repeating unit of chromatin is the nucleosome core, which consists of 147 base pairs of DNA wrapped in 1.7 left-handed superhelical turns around the surface of an octameric protein core formed by two molecules each of histones H2A, H2B, H3, and H4. Histones are highly basic proteins that bind very avidly and non-specifically to nucleic acids. Histones were among the first proteins studied due to their relative ease of isolation and all four histone proteins (H2A, H2B, H3, and H4) can be expressed in bacteria. This has allowed purifying and reconstituting of the histone proteins in cell free systems using well defined protocols. Though the native histone proteins undergo an extensive array of posttranslational modifications, recombinant histones do not undergo posttranslational modifications and can be obtained in a highly pure form due to their high expression levels.
Single Strand DNA-Binding Proteins (hereinafter referred to as “SSBP”) are ubiquitously expressed and involved in a variety of DNA metabolic processes including replication, recombination, damage, and repair. SSBP-1 is a housekeeping gene involved in mitochondrial biogenesis. It is also a subunit of a single-stranded DNA (ssDNA)-binding complex involved in the maintenance of genome stability.
Ribonuclease III (hereinafter referred to as “RNase III”) is an enzyme that is expressed in most of the cells and is involved in the processing of pre-rRNA. It has a catalytic domain and an RNA binding domain that is located in the C-terminal end of the enzyme. Inhibition of human RNase III resulted in cell death suggesting a very important role of this enzyme.
Gene therapy and exon skipping have served as a means of gene transduction or gene manipulation respectively in humans during the past two decades. Gene therapy and exon skipping were initially developed as therapeutic strategies focused to address detrimental monogenetic diseases for which there were no available options for treatment, e.g. primary immunodeficiency. These approaches later found widespread application in curing neurodegenerative diseases, cancer, metabolic disorders, and more.
Gene therapy involves delivery of genes of interest cloned in viral vectors which are capable of producing viruses when transduced in human cells. Despite the continuous improvement of retroviral and lentiviral gene transfer systems for gene delivery during the last many years, there remain severe limitations preventing the development of efficient and safe clinical applications for these systems. These limitations include: their inability to target infection to cells of interest, inefficient transduction, propensity of viral vectors to get incorporated in human genome and create mutations, elicited high immune responses, inability to be administered intravenously or subcutaneously, and intramuscular administration that only leads to local delivery of the gene. Owing to these limitations, no gene therapy based medication has been approved by FDA for use in humans, though there have been many clinical trials during the past two decades and also many ongoing clinical trials.
Exon skipping is a therapeutic strategy where antisense oligonucleotides (AO) are delivered in humans to modulate splicing of genes resulting in mRNA that either produces functional proteins or blocks their production. AOs are short nucleic acid sequences designed to selectively bind to specific mRNA or pre-mRNA sequences. Despite the very convincing underlying principle behind this strategy, only one AO has been approved by the FDA (Vitravene™, an intraocular injection to inhibit cytomegalovirus retinitis in immunocompromised patients; Isis Pharmaceuticals, Carlsbad, Calif.), and this drug is no longer marketed. There are certain limitations associated with the use of AOs including difficulty in achieving pharmacologically significant concentrations in cells due to biological barriers like endothelial and basement membrane, cell membrane, and sequestration by phagolysosomes.
Further discussion on the subjects of gene transfer and delivery may be found in U.S. Pat. No. 7,531,647 (“Lentiviral Vectors for Site-Specific Gene Insertion”); U.S. Pat. No. 8,158,827 (“Transfection Reagents”); and U.S. Pat. No. 8,652,460 (“Gene Delivery System and Method of Use”) and U.S. patent application Ser. No. 14/635,012 (“Chimeric Dystrophin-VSV-G Protein to Treat Dystrophinopathies”. The disclosures of each of U.S. Pat. Nos. 7,531,647, 8,158,827 and 8,652,460 and U.S. application Ser. No. 14/635,012 are incorporated by reference herein in their entireties.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects and advantages of the invention will be apparent from the description and drawings, and from the claims.